Project Summary Advances in genomic technologies have accelerated the pace of discovery with respect to genetic variation. The use of model systems is an expeditious route for the assessment of distinctions that render select individuals in a population more resilient to cellular stress and dysfunction. We have developed in vivo assays using the nematode, Caenorhabditis elegans, that facilitate isolation of conserved modulators of dopamine (DA) neurodegeneration and Parkinson?s disease (PD). To discern molecular factors conferring ?resilience?, it is essential to define our experimental goals in practical terms. Here, an inherent capacity of DA neurons to resist progressive degeneration induced by a-synuclein, a protein central to PD, represents an operational definition of resilience. C. elegans facilitates rapid analysis among isogenic populations and highly precise quantitation of neurodegeneration at a single-neuron level in animals challenged by a-synuclein. In Aim I, we will test a hypothesis for the mechanistic nature of dopaminergic resilience that postulates the system by which small RNA transport for epigenetic transmission intersects with regulators of DA transport and endocytosis to provide neuroprotection. A recently identified gene found to contain exonic variation in PD patients, TNK2, encodes a protein that controls dopamine transporter endocytosis. Intriguingly, the sole worm ortholog of TNK2 is SID-3, an endocytic regulator of the SID-1 dsRNA transporter responsible for systemic RNAi. Preliminary data demonstrate that TNK2/SID-3 modulates neurodegeneration. Moreover, we reported the discovery of a neuroprotective small molecule that acts directly on an E3 ligase, Nedd4, responsible for targeting TNK2 for degradation. Using mutant and transgenic analysis, we will dissect the role of Nedd4 in modulating both DA and small RNA transport via TNK2/SID-3. Since we hypothesize resilience is mediated through the activity of select miRNAs, in Aim 2, as a complementary approach, we will evaluate a set of epigenetically-regulated gene targets of a specific miRNA, mir-2 for DA neuroprotection. We have discerned that genomic knockout of mir-2 confers robust protection from a-synuclein-induced DA neurodegeneration in transgenic C. elegans. Therefore, we will conduct a systematic functional analysis of a subset of prioritized candidate gene targets predicted to be regulated by mir-2. Animals will be rigorously examined to quantify functional effects on DA neuron degeneration by conditional knockdown using DA neuron-specific RNAi. We will then counterscreen mir-2 targets to categorize either cell autonomous or non-cell autonomous effects on neuroprotection. We will subsequently coordinate our results with human genomic datasets to identify any single-nucleotide polymorphisms in candidate genes, and functionally evaluate conserved variations. Positives will be advanced for behavioral, lifespan and healthspan analyses. These assays provide excellent training opportunities for undergraduates who routinely conduct such methods in our lab. In all, our strategy is designed as an integrated approach towards expanding new therapeutic targets and mechanisms to halt neurodegeneration.